Aptamer sensor based on bimetal organic framework nano-enzyme as well as preparation method and application of aptamer sensor

Abstract

The invention discloses an aptamer sensor based on a bimetallic organic framework nano-enzyme as well as a preparation method and application of the aptamer sensor. According to the aptamer sensor, the catalytic activity of the bimetallic organic framework nano-enzyme is simply and conveniently regulated through an aptamer, so that colorimetric and photo-thermal dual-mode detection on kanamycin is realized; the aptamer can regulate and control the activity of the bimetal organic framework nano-enzyme through co-incubation without immobilization, the aptamer falls off from the surface of the nano-enzyme to generate signal response by utilizing the high selective affinity of kanamycin to the aptamer, and a temperature signal is generated by further utilizing the photothermal effect of an oxidation product. Therefore, the comparison of color and photo-thermal double-signal detection results is realized, and the credibility of the detection results is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of aptamer sensors, and in particular to an aptamer sensor based on a bimetallic organic framework nanozyme and a preparation method and application thereof. Background Art

[0002] Kanamycin (KAN) is an aminoglycoside antibiotic extracted from Streptomyces kanamycinus with broad-spectrum antibacterial properties that inhibit the growth and proliferation of microorganisms. KAN is widely used in agriculture, animal husbandry, and aquaculture to treat bacterial infections. However, overuse or abuse of KAN can lead to excessive biological residues in the environment and food, and produce other dangerous side effects on the human body, such as hearing defects, kidney damage, and anaphylactic shock. Therefore, accurate detection of KAN is of great significance to ensure food safety and human health. Traditional KAN detection methods include liquid chromatography-tandem mass spectrometry, high-performance liquid chromatography, and high-performance capillary electrophoresis, but these methods are often complex and time-consuming to operate, require expensive and bulky instruments, and rely on professional technicians, making them difficult to popularize in real-time detection samples. Therefore, it is necessary to develop low-cost, easy-to-operate sensors to meet the needs of food safety and customs supervision.

[0003] In recent years, aptamer-based biosensors (i.e., aptamer sensors) have been widely used in the field of KAN detection. However, in most cases, aptamers must be coupled to biosensors to obtain specific and reliable binding reactions, resulting in their detection performance being heavily dependent on the efficiency of biomolecule immobilization. Therefore, the development of aptamer sensors with simple signal output without immobilization is crucial for efficient detection of KAN. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide an aptamer sensor based on a bimetallic organic framework nanozyme and a preparation method and application thereof. The aptamer sensor based on a bimetallic organic framework nanozyme provided by the present invention can be used for colorimetric and photothermal dual-mode detection of kanamycin. The aptamer can regulate the activity of the bimetallic organic framework nanozyme by co-incubation without immobilization. The high selective affinity of kanamycin to the aptamer is utilized to make the aptamer fall off the surface of the nanozyme to generate a signal response. The temperature signal generated by the photothermal effect of the oxidation product is further utilized to realize the comparison of the colorimetric and photothermal dual signal detection results, thereby improving the credibility of the detection results.

[0005] The above object of the present invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention is a method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme, comprising the following steps:

[0007] (1) dissolving iron salt, zirconium acetate, ligand and acidity regulator in a solvent, and obtaining a bimetallic organic framework nanozyme by a hydrothermal reaction;

[0008] (2) Mixing the bimetallic organic framework nanozyme solution and the kanamycin aptamer (KAN-Aptamer, abbreviated as KAN-Apt) solution, and obtaining the bimetallic organic framework nanozyme-based aptamer sensor by incubation.

[0009] Furthermore, in step (1), the iron salt is ferric chloride.

[0010] Furthermore, in step (1), the ligand is 2-aminoterephthalic acid.

[0011] Furthermore, in step (1), the acidity regulator is acetic acid.

[0012] Furthermore, in step (1), the solvent is N,N-dimethylformamide.

[0013] Replacing the metal ion source will affect the shape and subsequent performance of the nanozyme. If zirconium acetate is replaced with zirconium chloride, the bimetallic organic framework nanozyme obtained by the reaction is a regular octahedron, and the addition of an aptamer will inhibit the enzyme activity. The bimetallic organic framework nanozyme prepared by the present invention is a biconical cylinder, and the addition of an aptamer can enhance the enzyme activity.

[0014] Furthermore, in step (1), the molar ratio of the iron salt, zirconium acetate and ligand is (0.5-1.5):(0.5-1.5):(1-3), preferably 1:1:2.

[0015] Furthermore, in step (1), the conditions of the hydrothermal method are: reacting at 110-120° C. for 10-12 hours.

[0016] Furthermore, in step (1), after the reaction is completed, a washing and drying step is also included.

[0017] Furthermore, in step (2), the bimetallic organic framework nanozyme is dissolved in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, CAS No. 7365-45-9) buffer to obtain a solution of the bimetallic organic framework nanozyme.

[0018] Furthermore, in step (2), the volume ratio of the bimetallic organic framework nanozyme solution to the kanamycin aptamer solution is (0.5-2):1, preferably 1:1.

[0019] Furthermore, in step (2), the concentration of the bimetallic organic framework nanozyme in the bimetallic organic framework nanozyme solution is 1-5 mg / mL, and the concentration of the kanamycin aptamer in the kanamycin aptamer solution is 5-20 μM.

[0020] Furthermore, in step (2), the sequence of the kanamycin aptamer is 5′-TGG GGG TTG AGG CTAAGC CGA-3′.

[0021] The second aspect of the present invention provides an aptamer sensor based on a bimetallic organic framework nanozyme obtained by the preparation method described in the first aspect.

[0022] The third aspect of the present invention provides the application of the aptamer sensor based on the bimetallic organic framework nanozyme described in the second aspect in the colorimetric and photothermal dual-mode detection of kanamycin.

[0023] Nanozymes are often combined with aptamers to develop aptamer sensors due to their excellent catalytic activity, good stability, and simple preparation. Among them, colorimetric methods based on nanozymes have attracted widespread attention due to their advantages such as obvious color change, convenient operation, low cost, and rapid naked eye observation. However, the sensitivity of a single colorimetric method may be easily interfered by environmental factors, resulting in inaccurate readings. Combining colorimetric methods with other sensing methods to build a multi-signal integrated platform may be a solution to greatly improve sensing performance. In contrast, multimodal methods can well inherit the visualization of the colorimetric mode while introducing multiple quantitative modes with different sensitivities to enhance the applicability of the overall detection. Among them, photothermal temperature readings as a complementary reading of colorimetric signals have attracted special attention because of their obvious advantages, such as easy-to-obtain results, reasonably priced thermometers, and low background signals. In addition, photothermal signals are not interfered by the background color of the sample and are applicable to a variety of food and environmental matrices. Therefore, the development of a dual-mode platform integrating colorimetric and photothermal readings is of great significance for KAN detection.

[0024] The fourth aspect of the present invention provides a method for colorimetric and photothermal dual-mode detection of kanamycin based on a bimetallic organic framework nanozyme aptamer sensor, comprising the following steps:

[0025] S1. Add the aptamer sensor based on the bimetallic organic framework nanozyme described in the second aspect, buffer, H 2 O 2 solution and 3,3',5,5'-tetramethylbenzidine solution, and after incubation, a kanamycin standard mixed solution is obtained;

[0026] S2. Determine the absorbance value of the kanamycin standard mixed solution by using an ultraviolet-visible spectrophotometer to establish a quantitative relationship between the absorbance value and the kanamycin concentration; irradiate the kanamycin standard mixed solution with a near-infrared (NIR) laser, and collect its temperature value by using a thermal imager to establish a quantitative relationship between the temperature value and the kanamycin concentration;

[0027] S3. Add the aptamer sensor based on the bimetallic organic framework nanozyme described in the second aspect, buffer, H 2 O 2 solution, 3,3',5,5'-tetramethylbenzidine solution, and incubating to obtain a mixed solution to be tested;

[0028] S4. Determine the absorbance value of the mixed solution to be tested by using an ultraviolet-visible spectrophotometer, and determine the kanamycin concentration in the test solution based on the quantitative relationship between the absorbance value and the kanamycin concentration; irradiate the mixed solution to be tested with a near-infrared laser, collect its temperature value by using a thermal imager, and determine the kanamycin concentration in the test solution based on the quantitative relationship between the temperature value and the kanamycin concentration.

[0029] Furthermore, in step S1, the kanamycin standard solution, the aptamer sensor based on the bimetallic organic framework nanozyme, and H 2 O 2 The volume ratio of the solution and 3,3',5,5'-tetramethylbenzidine (TMB) solution is (15-25):(35-45):(15-25):(90-110).

[0030] Furthermore, in step S1, the buffer solution is acetic acid-sodium acetate buffer solution.

[0031] Furthermore, in step S1, the volume ratio of the aptamer sensor based on the bimetallic organic framework nanozyme to the buffer solution is (35-45):(750-850).

[0032] Further, in step S1, the H 2 O 2 The concentration of the solution is 25-200 mM.

[0033] Furthermore, in step S1, the concentration of the TMB solution is 2-12 mM.

[0034] Furthermore, in step S2, a quantitative relationship between the absorbance value of the kanamycin standard mixed solution at 652 nm and the kanamycin concentration is established.

[0035] Furthermore, in step S3, the test liquid, the aptamer sensor based on the bimetallic organic framework nanozyme, H 2 O 2The volume ratio of solution and TMB solution is (15-25):(35-45):(15-25):(90-110).

[0036] Furthermore, in step S3, the buffer solution is acetic acid-sodium acetate buffer solution.

[0037] Furthermore, in step S3, the volume ratio of the bimetallic organic framework nanozyme-based aptamer sensor to the buffer solution is (35-45):(750-850).

[0038] Further, in step S3, the H 2 O 2 The concentration of the solution is 25-200 mM.

[0039] Furthermore, in step S3, the concentration of the TMB solution is 2-12 mM.

[0040] Furthermore, in steps S2 and S4, 808 nm near-infrared laser is used for irradiation.

[0041] Beneficial effects of the present invention:

[0042] The present invention realizes colorimetric and photothermal dual-mode detection of kanamycin by simply regulating the catalytic activity of the bimetallic organic framework nanozyme through aptamers. Firstly, 2-aminoterephthalic acid is used as a ligand, and iron salt and zirconium salt are used as metal nodes. A biconical cylindrical Zr-Fe bimetallic organic framework nanozyme (Zr-Fe-MOF) is prepared by a simple one-pot hydrothermal method. The prepared Zr-Fe-MOF has excellent enzyme-like catalytic activity and can be used in H 2 O 2 In the presence of , it catalyzes the oxidation of colorless TMB to generate a blue product, generating a colorimetric signal. At the same time, the oxidation product has a photothermal effect and can generate a temperature signal. Secondly, the introduction of the aptamer for simple co-incubation can significantly improve the catalytic activity of Zr-Fe-MOF. Then, the target kanamycin has a highly selective affinity for the aptamer, causing the aptamer to fall off the surface of Zr-Fe-MOF, thereby reducing its enzyme catalytic activity, resulting in a decrease in both absorbance and temperature. With kanamycin concentration as the horizontal coordinate and ultraviolet absorbance and temperature as the vertical coordinates, a standard curve for kanamycin detection is drawn to achieve colorimetric and photothermal dual-mode detection of kanamycin. The dual-mode detection results can be calibrated with each other to improve the credibility of the detection results; and the corresponding detection method can be selected according to actual conditions, making the constructed aptamer sensor more flexible. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the SEM image of the Zr-Fe-MOF prepared in Example 1.

[0044] Figure 2 The test results of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 are shown in Figure 1; wherein, (A) is the ultraviolet-visible absorption spectrum (UV-vis) of Zr-Fe-MOF and Zr-Fe-MOF / KAN-Apt, (B) is the Zeta potential diagram of TMB, Zr-Fe-MOF, Zr-Fe-MOF / KAN-Apt and after adding KAN, (C) is the UV-vis absorption spectrum of Zr-Fe-MOF and Zr-Fe-MOF / KAN-Apt to ABTS, and (D) is the test result diagram of the catalytic activity of Zr-Fe-MOF in the presence of different charged polymers.

[0045] Figure 3 Schematic diagram of the colorimetric and photothermal dual-mode detection of kanamycin by the aptamer sensor based on the bimetallic organic framework nanozyme provided by the present invention.

[0046] Figure 4 This is a diagram showing the optimization of detection conditions for the method of colorimetric and photothermal dual-mode detection of kanamycin based on the aptamer sensor of the bimetallic organic framework nanozyme in Example 5; wherein (A) is the concentration of KAN-Apt, and (B) is the H 2 O 2 concentration, (C) is TMB concentration, (D) is aptamer adsorption time, (E) is target (kanamycin) and KAN-Apt binding time, (F) is chromogenic substrate catalytic color development time, and the error bars represent the relative standard deviation of three independent measurements.

[0047] Figure 5 The figures are the detection result diagrams of colorimetric and photothermal dual-mode detection of kanamycin by aptamer sensor based on bimetallic organic framework nanozyme; wherein, (A) is the data diagram of absorbance value of kanamycin standard mixed solution measured by UV-visible spectrophotometer in Example 6, and the inset is the actual picture under daylight at the corresponding concentration; (B) is the relationship diagram between kanamycin concentration and absorbance value at 652nm; (C) is the standard curve diagram of kanamycin concentration in the range of 0 to 10μM and absorbance value at 652nm; (D) is the standard curve diagram of kanamycin concentration in the range of 0 to 10μM and temperature value, and the inset is the image captured by the corresponding infrared camera at different concentrations.

[0048] Figure 6The performance evaluation diagram of the colorimetric and photothermal dual-mode detection of kanamycin by the Zr-Fe-MOF-based aptamer sensor; wherein, (A) is a data diagram of the ultraviolet absorbance value at 652nm when the Zr-Fe-MOF-based aptamer sensor coexists with different antibiotics and kanamycin, (B) is a data diagram of the corresponding absorbance value of 8 parallel samples with kanamycin concentrations of 2 and 8 μM detected by two batches of Zr-Fe-MOF aptamer sensors, respectively, (C) is a data comparison diagram of the average result in (B), and the error bars represent the relative standard deviation of three independent measurements. DETAILED DESCRIPTION

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0050] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0052] Example 1

[0053] A method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme (Zr-Fe-MOF), comprising the following steps:

[0054] (1) Weigh 360 mg of ferric chloride hexahydrate, 164.3 mg of zirconium acetate and 483 mg of 2-aminoterephthalic acid and add them to 25 mL of N,N-dimethylformamide, then add 5 mL of glacial acetic acid and stir magnetically for 30 min to ensure full dissolution. The mixture was then transferred to a 50 mL autoclave and thermally reacted at 120 ° C for 12 hours. After the reaction was completed, it was cooled to room temperature, and the reaction product was washed 4 times by centrifugation with methanol and ethanol (8000 rpm, 10 min), and finally vacuum dried at 60 ° C overnight to obtain a bimetallic organic framework nanozyme (Zr-Fe-MOF).

[0055] (2) Zr-Fe-MOF was dissolved in HEPES buffer (0.1 M, pH = 5.4) to obtain a 1 mg / mL Zr-Fe-MOF solution; kanamycin aptamer (KAN-Apt) was dissolved in HEPES buffer (0.1 M, pH = 5.4) to obtain a 10 μM KAN-Apt solution; 20 μL of KAN-Apt solution (10 μM) was added to 20 μL of Zr-Fe-MOF solution (1 mg / mL), the sequence of KAN-Apt was 5′-TGG GGG TTG AGG CTA AGC CGA-3′, the mixture was mixed and incubated at room temperature for 5 min (aptamer adsorption time) to obtain an aptamer sensor based on Zr-Fe-MOF (Zr-Fe-MOF / KAN-Apt).

[0056] Figure 1 is a scanning electron microscope (SEM) image of the Zr-Fe-MOF prepared in Example 1. Figure 1 It can be seen that Zr-Fe-MOF is a biconical cylinder with a length of about 900nm and a width of about 250nm.

[0057] like Figure 2 As shown in (A), after incubation of Zr-Fe-MOF and KAN-Apt, there is an obvious absorption peak at 260nm, which is the UV absorption peak of KAN-Apt, indicating that KAN-Apt is bound to Zr-Fe-MOF. Further, the Zeta potential was tested to explore the mechanism of aptamer regulating nanozyme activity, such as Figure 2 As shown in (B), TMB has two amino groups, so the dissociation constant (pKa) of non-oxidized TMB is about 4.2, making the zeta potential of TMB positive at pH = 4.0. At the same time, Zr-Fe-MOF is also positively charged, +7.92±0.46mV, but after combining with KAN-Apt, the complex Zr-Fe-MOF / KAN-Apt becomes negatively charged, -14.20±1.32mV, which is due to the negative charge of the KAN-Apt phosphate skeleton. The negative charge of the complex will enhance the affinity for the positively charged TMB, resulting in enhanced catalytic activity for TMB. After further addition of the target KAN, the charge of Zr-Fe-MOF / KAN-Apt becomes more positive, -5.66±0.14mV, indicating that KAN-Apt falls off the surface of Zr-Fe-MOF. After replacing the positively charged TMB with the negatively charged 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS, CAS No. 30931-67-0), the catalytic activity of Zr-Fe-MOF / KAN-Apt towards ABTS decreased significantly, e.g. Figure 2As shown in (C), this is due to the electrostatic repulsion between ABTS and Zr-Fe-MOF / KAN-Apt. The above results indicate that the electrostatic interaction between the substrate and the surface-modified Zr-Fe-MOF has a great influence on the catalytic activity of Zr-Fe-MOF.

[0058] This conclusion can be verified by testing the catalytic activity of Zr-Fe-MOF in the presence of polymers with different charges, such as Figure 2 As shown in (D), the enzyme catalytic activity of Zr-Fe-MOF was significantly enhanced when it was combined with negatively charged polymers such as polystyrene sulfonate (PSS) and KAN-Apt compared to the untreated Zr-Fe-MOF in the control group. In contrast, the enzyme catalytic activity decreased and remained unchanged when combined with positively charged polyethyleneimine (PEI) and uncharged polyvinylpolypyrrolidone (PVP). These results indicate that only negatively charged polymers can enhance the catalytic activity of Zr-Fe-MOF, and KAN-Apt has the best effect.

[0059] Example 2

[0060] A method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme (Zr-Fe-MOF) is basically the same as that in Example 1, except that in step (2), the concentration of the KAN-Apt solution is 5 μM.

[0061] Example 3

[0062] A method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme (Zr-Fe-MOF) is basically the same as that in Example 1, except that in step (2), the concentration of the KAN-Apt solution is 15 μM.

[0063] Example 4

[0064] A method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme (Zr-Fe-MOF) is basically the same as that in Example 1, except that in step (2), the concentration of the KAN-Apt solution is 20 μM.

[0065] Example 5

[0066] A method for colorimetric and photothermal dual-mode detection of kanamycin based on a bimetallic organic framework nanozyme aptamer sensor comprises the following steps:

[0067] S1. Add 40 μL of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 to 20 μL of kanamycin solution (2 μM), incubate for 15 min (target binding time), then add 820 μL of acetic acid-sodium acetate buffer (0.2 M, pH = 4.0), 20 μL of H2 O 2 solution and 100 μL TMB solution, and incubate for 6 min (catalytic color development time) to obtain a kanamycin mixed solution;

[0068] S2. Determine the absorbance value of the kanamycin mixed solution using an ultraviolet-visible spectrophotometer; irradiate the kanamycin mixed solution with an 808 laser (1 W) for 10 min, and collect its temperature value using a thermal imager.

[0069] The principle diagram of the colorimetric and photothermal dual-mode detection of kanamycin by the aptamer sensor based on the bimetallic organic framework nanozyme provided by the present invention is as follows: Figure 3 shown.

[0070] Investigating the KAN-Apt concentration (Examples 1-4) and H 2 O 2 The concentration of TMB and the aptamer adsorption time (the time for the nanozyme and the aptamer to be mixed and incubated in step (2) of Example 1), the target binding time (the time for the target to bind to the aptamer after adding the target to the sensor) and the catalytic color development time (the time for the target to bind to the aptamer after adding H 2 O 2 The effect of the time from the start of catalytic color change after the solution and TMB solution to the test on the detection of kanamycin, the test results are as follows Figure 4 As shown in the figure, the optimal experimental conditions of this method are as follows: KAN-Apt concentration is 10 μM, H 2 O 2 The concentration was 100 mM, the TMB concentration was 10 mM, the aptamer adsorption time was 5 min, the target binding time was 15 min, and the catalytic color development time was 6 min.

[0071] Example 6

[0072] A method for colorimetric and photothermal dual-mode detection of kanamycin based on a bimetallic organic framework nanozyme aptamer sensor, establishing a standard curve for colorimetric and photothermal dual-mode detection of kanamycin, comprising the following steps:

[0073] S1. Add 40 μL of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 to 20 μL of kanamycin standard solution (0 μM, 0.8 μM, 1.25 μM, 2.5 μM, 4 μM, 5 μM, 6 μM, 8 μM, 10 μM, 15 μM, 20 μM), incubate for 15 min, then add 820 μL of acetic acid-sodium acetate buffer (0.2 M, pH = 4.0), 20 μL of H 2 O 2solution (100 mM) and 100 μL TMB solution (10 mM) and mix well, and continue incubating at room temperature for 6 min to obtain a kanamycin standard mixed solution;

[0074] S2. Use a UV-visible spectrophotometer to measure the absorbance value of the kanamycin standard mixed solution, use the kanamycin concentration as the horizontal coordinate, and the absorbance value at 652nm as the vertical coordinate to establish a standard curve for colorimetric detection of kanamycin; use an 808nm near-infrared laser (1W) to irradiate the kanamycin standard mixed solution for 10 minutes, then use an infrared photothermal camera to take a picture and record the temperature value, use the kanamycin concentration as the horizontal coordinate, and the measured temperature value as the vertical coordinate to establish a standard curve for photothermal detection of kanamycin.

[0075] Example 6 The absorbance of kanamycin standard mixed solution was measured by UV-visible spectrophotometer. The illustration is a real picture of the corresponding concentration under sunlight. Figure 5 As shown in (A); under the optimal experimental conditions, as the concentration of kanamycin increases, the absorbance value at 652nm gradually decreases, as shown in Figure 5 As shown in (B); Figure 5 As shown in (C), the kanamycin concentration in the range of 0 to 10 μM has a linear relationship with the absorbance value at 652 nm, and the linear equation is: 652 =-0.03749C+0.98462, R 2 =0.99828, and the detection limit is 0.17 μM.

[0076] like Figure 5 As shown in (D), under the optimal experimental conditions, as the kanamycin concentration increases, the temperature value gradually decreases, and the temperature value has a good linear relationship with the kanamycin concentration in the range of 0 to 10 μM. The linear equation is: T = -0.52692C + 50.05839, R 2 =0.99143, and the detection limit is 0.47 μM.

[0077] Test Example 1

[0078] The specificity and anti-interference of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 to KAN were tested. The specificity was tested by adding 40 μL of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 to 20 μL of 5 μM kanamycin solution, 50 μM streptomycin (STR) solution, 50 μM chloramphenicol (CAP) solution, 50 μM tetracycline hydrochloride (TOC) solution, 50 μM neomycin (NEO) solution, 50 μM gentamicin (GNE) solution, and 50 μM oxytetracycline (OXY) solution, respectively. After incubation for 15 min, 820 μL of acetic acid-sodium acetate buffer (0.2 M, pH = 4.0) and 20 μL of H 2 O 2 solution (100 mM) and 100 μL of TMB solution (10 mM) were mixed and incubated for 6 min at room temperature to obtain a mixed solution. The absorbance of the mixed solution at 652 nm was measured using a UV-visible spectrophotometer.

[0079] The method for testing the anti-interference property is as follows: 40 μL of the Zr-Fe-MOF-based aptamer sensor prepared in Example 1 was added to 20 μL of 5 μM kanamycin solution, 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM STR solution, 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM CAP solution, 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM TOC solution, 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM NEO solution, 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM GNE solution, and 20 μL of 5 μM kanamycin solution + 20 μL of 50 μM OXY solution, respectively, and after incubation for 15 min, 800 μL of acetic acid-sodium acetate buffer (0.2 M, pH = 4.0) and 20 μL of H 2 O 2 solution (100 mM) and 100 μL of TMB solution (10 mM) were mixed and incubated for 6 min at room temperature to obtain a mixed solution. The absorbance of the mixed solution at 652 nm was measured using a UV-visible spectrophotometer.

[0080] The test results are as follows Figure 6 As shown in (A), the absorbance value of the Zr-Fe-MOF-based aptamer sensor will decrease significantly only when kanamycin is present, while there is no obvious change when other antibiotics exist alone, indicating that the sensor has excellent selectivity for kanamycin; after the coexistence of other antibiotics and kanamycin, the absorbance values ​​of the mixed solution are significantly reduced, indicating that the sensor has excellent anti-interference ability.

[0081] Test Example 2

[0082] The repeatability of the colorimetric and photothermal dual-mode detection of kanamycin by the aptamer sensor based on the bimetallic organic framework nanozyme was tested. The test method was as follows: two batches of Zr-Fe-MOF-based aptamer sensors were prepared according to the method of Example 1, and kanamycin solutions with concentrations of 2 μM and 8 μM were added under the optimal experimental conditions, respectively, and 8 parallel tests were performed for each concentration.

[0083] The test results are as follows Figure 6 As shown in (B), in one batch of Zr-Fe-MOF-based aptamer sensors, the relative standard deviations (RSDs) of the absorbance values ​​of 2μM and 8μM kanamycin solutions were 4.80% and 4.87%, respectively, while the results of another batch were 3.61% and 2.72%. The test results show that the aptamer sensors prepared based on Zr-Fe-MOF have good repeatability in the same batch.

[0084] Comparing the test results of two batches, such as Figure 6 As shown in (C), there is no statistically significant difference in the absorbance values ​​of 2μM and 8μM kanamycin solutions detected by the Zr-Fe-MOF-based aptamer sensor, indicating that the detection results of sensors from different batches are highly consistent, further demonstrating that the sensor has good repeatability.

[0085] Test Example 3

[0086] In order to verify the practical application of the method for colorimetric and photothermal dual-mode detection of kanamycin by the aptamer sensor based on the dual metal organic framework nanozyme of the present invention, different concentrations of kanamycin (0, 0.6 μM, 2.5 μM) were added to milk and honey. The above actual samples were respectively determined by the colorimetric method and photothermal method of the aptamer sensor based on Zr-Fe-MOF prepared in Example 1 and the national standard liquid chromatography-tandem mass spectrometry (LC-MS-MS). The results are shown in Table 1:

[0087] Table 1

[0088]

[0089] As can be seen from Table 1, the recovery rate of the colorimetric detection method is 98.34-108.80%, RSD≤8.47%; the recovery rate of the photothermal detection method is 91.67-106.80%, RSD≤10.46%. The test results show that the Zr-Fe-MOF-based aptamer sensor constructed by the present invention has good applicability.

[0090] In addition, there was no significant difference between the test results of the colorimetric and photothermal detection method and the national standard method (p>0.05), indicating that the constructed colorimetric and photothermal detection method can be used to detect kanamycin in actual samples, providing a reliable alternative method for the detection of kanamycin.

[0091] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. It should be understood by those skilled in the art that other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for preparing an aptamer sensor based on a bimetallic organic framework nanozyme, characterized in that: The following steps are involved: (1) dissolving iron salt, zirconium acetate, ligand and acidity regulator in a solvent, and obtaining a bimetallic organic framework nanozyme by a hydrothermal reaction; (2) Mixing the solution of the bimetallic organic framework nanozyme and the kanamycin aptamer solution, and obtaining the bimetallic organic framework nanozyme-based aptamer sensor by incubation.

2. The preparation method according to claim 1, characterized in that: In step (1), the ligand is 2-aminoterephthalic acid.

3. The preparation method according to claim 1, characterized in that: In step (1), the molar ratio of the iron salt, zirconium acetate and ligand is (0.5-1.5):(0.5-1.5):(1-3).

4. The preparation method according to claim 1, characterized in that: In step (2), the volume ratio of the bimetallic organic framework nanozyme solution to the kanamycin aptamer solution is (0.5-2):

1.

5. The preparation method according to claim 4, characterized in that: In step (2), the concentration of the bimetallic organic framework nanozyme in the bimetallic organic framework nanozyme solution is 1-5 mg / mL, and the concentration of the kanamycin aptamer in the kanamycin aptamer solution is 5-20 μM.

6. The preparation method according to claim 1, characterized in that: In step (2), the sequence of the kanamycin aptamer is 5′-TGG GGG TTG AGG CTA AGC CGA-3′.

7. An aptamer sensor based on a bimetallic organic framework nanozyme obtained by the preparation method described in any one of claims 1 to 6.

8. Application of the bimetallic organic framework nanozyme-based aptamer sensor according to claim 7 in colorimetric and photothermal dual-mode detection of kanamycin.

9. A method for colorimetric and photothermal dual-mode detection of kanamycin based on a bimetallic organic framework nanozyme aptamer sensor, characterized in that: The following steps are involved: S1. Add the bimetallic organic framework nanozyme-based aptamer sensor according to claim 7, buffer, H2O2 solution and 3,3',5,5'-tetramethylbenzidine solution to the kanamycin standard solution, and incubate to obtain a kanamycin standard mixed solution; S2. Determine the absorbance value of the kanamycin standard mixed solution by using an ultraviolet-visible spectrophotometer to establish a quantitative relationship between the absorbance value and the kanamycin concentration; irradiate the kanamycin standard mixed solution with a near-infrared laser, and collect its temperature value by using a thermal imager to establish a quantitative relationship between the temperature value and the kanamycin concentration; S3. Add the bimetallic organic framework nanozyme-based aptamer sensor according to claim 7, buffer, H2O2 solution, and 3,3',5,5'-tetramethylbenzidine solution to the test solution, and incubate to obtain a mixed solution to be tested; S4. Determine the absorbance value of the mixed solution to be tested by using an ultraviolet-visible spectrophotometer, and determine the kanamycin concentration in the test solution based on the quantitative relationship between the absorbance value and the kanamycin concentration; irradiate the mixed solution to be tested with a near-infrared laser, collect its temperature value by using a thermal imager, and determine the kanamycin concentration in the test solution based on the quantitative relationship between the temperature value and the kanamycin concentration.

10. The method according to claim 9, characterized in that In step S1, the volume ratio of the kanamycin standard solution, the bimetallic organic framework nanozyme-based aptamer sensor, the H2O2 solution and the 3,3',5,5'-tetramethylbenzidine solution is (15-25):(35-45):(15-25):(90-110).

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